The present invention relates to certain sugars. More specifically, this invention provides glycosylated polyamine compounds, methods for their synthesis, characterization, their use as ligands in preparing metal complexes for developing analytical methodology, and their pharmaceutical uses, for example, as antitumor agents or modulators of estrogen receptor activity.
Glycosylated amines, also variously known as N-glycosides, glycosylamines, or aminoglycosides, are formed by reacting a carbonyl containing sugar molecule with an amine. Glycosylated amines are known in the fields of polymer chemistry, and cosmetics. For example, glycosylated amines from primary amines of intermediate molecular weight have been reported to be good wetting agents. Mitts, E. and Hixon, R. M., J. Am. Chem. Soc., 66: 483 (1944). Glycosylated amines as a class have been reported as components for detergents and cosmetics, surfactants, polymers, sweeteners and as liquid crystalline compounds. Lammers, et al., Tetrahedron, 59: 8103 (1994). Glycosylamines also have been used in kraft pulping liquor in the wood processing industry. MacLeod, J. M., Carbohydrate Res., 75: 71 (1979).
Glycosylated amines also play a vital role at the cellular level, because they are essential components of nucleic acids, wherein the ring nitrogen atoms of purine or pyrimidine bases form N-glycosyl linkages with carbon atom 1 of D-ribose or 2-deoxy-D-ribose, which are incorporated into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), respectively. While the physiological functions of some of the glycosylated amines appear to have been examined, their potential as pharmacological agents has not been fully explored.
Lammers et al. supra, have disclosed the preparation of mono- and diglycosylamines, wherein the amine and the saccharide were mixed in water under reductive amination conditions. However the yields were poor. Mitts et al. supra, reported the preparation of N,Nxe2x80x2-propylenediglucamine by refluxing glucose with propylenediamine in methanol followed by reduction over activated Raney Nickel and at high pressures and temperatures. The yield appeared to be very low in this case also because it was reported that only a very small amount of the reduced compound was isolated. Mitts et al. further disclosed that attempts to isolate and characterize the condensation products of amines such as isopropylamine, 2-aminooctane, and propylenediamine with glucose were not successful. Accordingly, there exists a need for a synthetic procedure to prepare glycosylated amines and glycosylated polyamines comprising a variety of sugars and amines.
Many analytical techniques have been developed to characterize glycosylamines. Only recently, however, has the research focussed on investigation of linkage information of metal cationized oligosaccharides by mass spectrometry (MS) and tandem mass spectrometry (MSn). Asam, M. R. and Glish, G. L., J. Am. Soc. Mass Spectrom., 8: 987 (1998); Weiskopf, et al. Rapid Com. Mass Spec., 11: 1493 (1997); Hofineister, et al., J. Am. Chem. Soc., 113: 5964 (1991); and Fura, A. and Leary, J. A., Anal. Chem., 65: 2805 (1993).
Mass spectrometry is not a tool traditionally used to distinguish stereoisomers. However, the stereochemistry of individual monosaccharides as well as xcex1 versus xcex2 configuration of glycosidic bonds in disaccharides can be determined by MSn. See for example, Gaucher, S. P. and Leary, J. A., Anal. Chem., 70: 3009 (1998); Smith, et al. J. Org. Chem., 62: 2152 (1997). This method involves cationizing the saccharide using a metal-ligand system such as Zn(diethylenetriamine)2Cl2 or Ni(1,3-diaminopropane)3Cl2, by allowing metal N-glycoside complexes to form in solution. Yano, S., Coord. Chem. Rev., 92: 113 (1988); and Yano, et al. J. Chem. Soc., Dalton Trans., 1699 (1993). The complexes are then transferred from solution to the gas phase by electrospray ionization (ESI) or fast atom bombardment (FAB) and analyzed by tandem mass spectrometry (MS/MS). See for example, Gaucher and Leary, supra; Smith and Leary, supra; and Smith, et al., supra. The axial versus equatorial stereochemistry of the C2 and C4 hydroxyl groups could be differentiated by the cross ring cleavage patterns observed in the gas phase.
In the above-described mass spectrometric procedures, it is time consuming to screen the efficacy of different metals for a given saccharide or ligand because each individual metal-ligand complex requires synthesis a priori. Accordingly, methods for rapid synthesis of metal-ligand complexes are needed so that the metal-ligand complexes so formed can be readily analyzed upon their synthesis.
All literature references, patents, and patent applications cited in this specification are hereby incorporated by reference in their entirety.
The present invention discloses glycosylated polyamines, methods for their preparation and use, and pharmaceutically acceptable compositions comprising gylcosylated polyamines. Glycosylated polyamines, in one embodiment, are prepared from two or more saccharides and a polyamine. In one embodiment, the glycosylated polyamine compound has the following formula (Formula I):
R1xe2x80x94Zxe2x80x94R2xe2x80x83xe2x80x83(Formula I)
wherein:
each of R1 and R2 is independently a monosaccharide residue or an oligosaccharide residue; Z is an aliphatic polyamino linker that is the residue of an aliphatic polyamine comprising at least two amino groups, each of which is independently a primary or secondary amino group; and each of R1 and R2 is linked through its anomeric carbon at its 1 position to a different amino group of the aliphatic polyamino linker to form a glycosidic bond;
provided that when each of R1 and R2 is the same and is a glucose, galactose, mannose, or cellobiose residue, Z is the residue of an aliphatic polyamine other than ethylenediamine or diaminopropane;
and pharmaceutically acceptable salts, prodrugs and derivatives thereof.
In one embodiment, each of R1 and R2 of Formula I is an oligosaccharide residue. In another embodiment, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine.
In a further embodiment, the group in equatorial conformation at C2 position is a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, aminothiol, amino alcohol, spermine, or nitro group, and optionally a hydrogen in the axial conformation.
In one embodiment of Formula I, R1 is a monosaccharide residue and R2 is an oligosaccharide residue. In another embodiment, each of R1 and R2 of Formula I is a monosaccharide residue. In yet another embodiment, each of R1 and R2 of Formula I is a hexose residue. In a further embodiment, each of the hexose residues is independently substituted by one or more of the following groups: a lower alkyl, lower alkoxy, acyl, carboxy, carboxyamino, amino, acetamido, halo, thio, or nitro; provided that the anomeric carbon has a free hydroxyl group to form a glycosidic linkage with the aliphatic polyamino linker.
In one embodiment, at least one of R1 and R2 has a group other than hydrogen in equatorial conformation at the C2 position that is adjacent to the anomeric carbon atom linked to the aliphatic polyamine. In another embodiment, the group in equatorial conformation at C2 position is a hydroxy, alkoxy, halo, lower alkyl, amino, N-acetyl, N-alkyl, N-hydroxy, N-alkoxy, or nitro group, and optionally a hydrogen in the axial conformation.
In one embodiment, the aliphatic polyamino linker of Formula I is a residue of diethylenetriamine. In a further embodiment, the diethylenetriamine residue is substituted by one or more the following groups: lower alkyl, hydroxy, lower alkoxy, amino, acyl, acetamido, halo, or nitro; provided that there is at least one amino group per each saccharide residue to form a glycosidic linkage.
Another embodiment presents a compound of Formula I, wherein each of R1 and R2 is the same and is a glucose, galactose, allose or fucose residue and the aliphatic polyamine linker is the residue of diethylenetriamine. Some specific embodiments include: diglucosyl-diethylenetriamine; digalactosyl-diethylenetriamine; diallosyl-diethylenetriamine; and difucosyl-diethylenetriamine; and their pharmaceutically acceptable salts, prodrugs and derivatives. One such salt may be HCl salt.
Pharmaceutically acceptable compositions comprising glycosylated polyamines, such as a compound of Formula I and pharmaceutically acceptable salts, prodrugs and derivatives thereof are also provided.
Glycosylated polyamine compounds, for example, of formula I, are effective as anticancer agents. Such anticancer activity may include inhibition of tumor cell growth, or multiplication or tumor size. Some examples of such compounds are diglycosylated diethylenetriamines, such as diglucosyl diethylenetriamine, digalactosyl diethylenetriamine, difucosyl diethylenetriamine, and diallosyl diethylenetriamine.
Accordingly, compounds of Formula I can be employed in methods to treat cancers or tumors. Such methods include providing one or more compounds of Formula I, their pharmaceutically effective salts, prodrugs and derivatives in an effective amount to treat a cancer or reduce the tumor cell growth or multiplication or tumor size. Exemplary cancers that may be treated include: leukemia, non-small-cell lung cancer, small-cell lung cancer, colon cancer, a cancer of the central nervous system, melanoma, ovarian cancer, breast cancer, renal and prostate cancer. The above-described compounds of Formula I can also be used in preparing one or medicaments to treat one or more of such cancers.
Furthermore, the compounds of the invention can be used as modulators of the estrogen receptor (ER), for example to modulate an estrogen receptor-mediated response. Based on crystallographic studies of their structure, compounds of the invention are predicted to interact with (bind to) the estrogen receptor and thus can be used to modulate ER activity, for example in a variety of conditions, disorders or diseases involving estrogen-mediated activities. In addition to treatment of hormonally-regulated cancers, such as breast cancers, such conditions, disorders and/or diseases include, for example, preventing, treating or delaying onset of osteoporosis, preventing or delaying onset of breast cancer in high risk women, modulation of ovulation (e.g., ovulation induction), preventing, treating or delaying onset of cardiovascular disorders (e.g., reduction of cholesterol levels), preventing, treating or delaying onset of uterine disorders (e.g., endometriosis, dysfunctional uterine bleeding, fibroids), and overall management of postmenopausal women""s health. A preferred compound for modulation of ER activity is diglucosyl diethylenetriamine (also referred to herein as SPG-20).
One specific embodiment of Formula I is diglucosyl diethylenetriamine. The diglucosyl diethylenetriamine can be prepared, for example, in a salt form such as HCl salt, which has the structure: 
Yet another specific embodiment of Formula I is digalactosyl diethylenetriamine. The digalactosyl diethylenetriamine can be prepared, for example, in a salt form, such as HCl salt, which has the structure: 
Another specific embodiment of Formula I is difucosyl diethylenetriamine. The difucosyl diethylenetriamine can be prepared, for example, in a salt form, such as HCl salt, which has the structure: 
Another specific embodiment of Formula I is diallosyl diethylenetriamine. The diallosyl diethylenetriamine can be prepared, for example, in a salt form such as HCl salt, which has the structure: 
The present invention also provides a metal-polyamine-N-glycosyl complex of Formula II:
[R1xe2x80x94Zxe2x80x94R2].Yxe2x80x83xe2x80x83(Formula II)
wherein in one embodiment, [R1xe2x80x94Zxe2x80x94R2] is represented by Formula I; and Y is a metal compound; and pharmaceutically acceptable salts, prodrugs and derivatives thereof.
In one embodiment, each of R1 and R2 is a hexose residue. In yet another embodiment, each of R1 and R2 is the same and is a glucose, galactose, allose or fucose residue.
In one embodiment of Formula II, Z is an aliphatic polyamino linker which is a residue of a polyamine selected from the group consisting of ethylene diamine, propylene diamine, diethylene triamine. In an embodiment of Formula II, the metal compound Y is a metal such as zinc, or a metal salt such as zinc chloride, zinc acetate, zinc triflate, sodium chloride magnesium chloride, copper chloride, cobalt chloride, nickel chloride, or calcium carbonate. In addition, the metal salts can be also those of organic origin such as sulfonate triflate or tosylate.
An embodiment of a glycosylated polyamine-zinc complex is N,Nxe2x80x2-dihexosyldiethylenetriamine-zinc chloride which has the following structure: 
In one embodiment, a method for preparing a metal-polyamine-N-glycosyl complex, for example, of Formula II:
[R1xe2x80x94Zxe2x80x94R2].Yxe2x80x83xe2x80x83Formula II
is provided which comprises:
a) providing a glycosylated polyamine compound, for example, of Formula I, [R1xe2x80x94Zxe2x80x94R2]; b) reacting the compound of Formula I with a metal compound, Y, and a salt, such as ammonium hydroxide, in a solvent, such as methanol, to form a metal complex having the formula, Formula II, [R1xe2x80x94Zxe2x80x94R2].Y; and, optionally, c) isolating the metal complex obtained in step b).
The metal compound Y and the compound of Formula I are preferably reacted in about equimolar amounts.
In one embodiment, the metal compound comprises a metal, such as zinc, or a metal salt, such as zinc chloride. An exemplary general reaction scheme to prepare a zinc chloride-diethylenetriamine-dihexosyl complex is shown below: 
The present invention also provides an analytical method to determine the stereospecificity of a glycosidic linkage between two sugars in a disaccharide. The method comprises the steps of: a) providing a glycosylated polyamine, for example, of Formula I, wherein at least one of R1 and R2 is a disaccharide; b) cationizing the glycosylated polyamine using a metal compound Y to form the corresponding metal-polyamine-N-glycoside complex of Formula II; c) ionizing the metal-polyamine-N-glycoside complex; and d) detecting the ions characteristic of a particular stereospecific linkage of the disaccharide using one or more mass spectrometers.
In one embodiment, the metal compound Y comprises zinc chloride or nickel chloride. In another embodiment, the metal-polyamine-glycoside complex is ionized through an electrospray ionization or a fast atom bombardment ionization technique. In yet another embodiment, the ion detection is accomplished by using two or more mass spectrometers arranged in tandem, in space and time, for example as triple quad instruments or those employing ion trap or ion cyclotron resonance technology.
The present invention also discloses a method for detecting the presence of an axial or equatorial conformation of a group, for example, at the C2 position of a saccharide, which method comprises the steps of: a) reacting an aliphatic polyamine and the saccharide in the presence of a precipitating agent; b) observing for a precipitate in the reaction mixture within a certain time, such as from within a few minutes to within a few hours, for example, 3-8 hours; and c) noting the presence of equatorial conformation of the group at the C2 position if a precipitate is observed in step b).
In one embodiment, the aliphatic polyamine and the saccharide are reacted at a certain ratio, for example, at about a 1:2 molar ratio. In another embodiment, the precipitating agent is present in about 1 molar concentration. In some embodiments, the precipitate can be observed within a few minutes, whereas in certain other embodiments, the precipitate is formed within a few hours; provided that the saccharide has an equatorial substitution at the C2 position.
In one embodiment, the substituent at the C2 position is a hydroxyl group. Other examples of substituents at the C2 position include alkoxy, halo, lower alkyl, amino, substituted amino, and nitro groups. The saccharide may be for example, a monosaccharide such as a pentose, or hexose, or an oligosaccharide. The precipitating agent can be any acidic salt that provides a halo counterion, for example, HCl, HBr, HI. Preferably, the counterion is a chloro ion. The aliphatic polyamine includes polyamines such as diethylene triamine, triethylenetetramine, ethylene diamine, and diaminopropane.